Inhibitors of IMPDH enzyme

ABSTRACT

The present invention relates to a novel class of compounds which inhibit IMPDH. This invention also relates to pharmaceutical compositions comprising these compounds. The compounds and pharmaceutical compositions of this invention are particularly well suited for inhibiting IMPDH enzyme activity and consequently, may be advantageously used as therapeutic agents for IMPDH mediated processes. This invention also relates to methods for inhibiting the activity of IMPDH using the compounds of this invention and related compounds.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a novel class of compounds whichinhibit IMPDH. This invention also relates to pharmaceuticalcompositions comprising these compounds. The compounds andpharmaceutical compositions of this invention are particularly wellsuited for inhibiting IMPDH enzyme activity and consequently, may beadvantageously used as therapeutic agents for IMPDH mediated processes.This invention also relates to methods for inhibiting the activity ofIMPDH using the compounds of this invention and related compounds.

BACKGROUND OF THE INVENTION

The synthesis of nucleotides in organisms is required for the cells inthose organisms to divide and replicate. Nucleotide synthesis in mammalsmay be achieved through one of two pathways: the de novo synthesispathway or the salvage pathway. Different cell types use these pathwaysto a different extent.

Inosine-5′-monophosphate dehydrogenase (IMPDH; EC 1.1.1.205) is anenzyme involved in the de novo synthesis of guanosine nucleotides. IMPDHcatalyzes the NAD-dependent oxidation of inosine-5′-monophosphate (IMP)to xanthosine-5′-monophosphate (XMP)[Jackson R. C. et. al., Nature, 256,pp. 331-333, (1975)].

IMPDH is ubiquitous in eukaryotes, bacteria and protozoa [Y. Natsumeda &S. F. Carr, Ann. N.Y. Acad., 696, pp. 88-93 (1993)]. The prokaryoticforms share 30-40% sequence identity with the human enzyme. Regardlessof species, the enzyme follows an ordered Bi-Bi reaction sequence ofsubstrate and cofactor binding and product release. First, IMP binds toIMPDH. This is followed by the binding of the cofactor NAD. The reducedcofactor, NADH, is then released from the product, followed by theproduct, XMP [S. F. Carr et al., J. Biol. Chem., 268, pp. 27286-90(1993); E. W. Holmes et al., Biochim. Biophys. Acta, 364, pp. 209-217(1974)]. This mechanism differs from that of most other knownNAD-dependent dehydrogenases, which have either a random order ofsubstrate addition or require NAD to bind before the substrate.

Two isoforms of human IMPDH, designated type I and type II, have beenidentified and sequenced [F. R. Collart and E. Huberman, J. Biol. Chem.,263, pp. 15769-15772, (1988); Y. Natsumeda et. al., J. Biol. Chem., 265,pp. 5292-5295, (1990)]. Each is 514 amino acids, and they share 84%sequence identity. Both IMPDH type I and type II form active tetramersin solution, with subunit molecular weights of 56 kDa [Y. Yamada et.al., Biochemistry, 27, pp. 2737-2745 (1988)].

The de novo synthesis of guanosine nucleotides, and thus the activity ofIMPDH, is particularly important in B and T-lymphocytes. These cellsdepend on the de novo, rather than salvage pathway to generatesufficient levels of nucleotides necessary to initiate a proliferativeresponse to mitogen or antigen [A. C. Allison et. al., Lancet II, 1179,(1975) and A. C. Allison et. al., Ciba Found. Symp., 48, 207, (1977)].Thus, IMPDH is an attractive target for selectively inhibiting theimmune system without also inhibiting the proliferation of other cells.

Immunosuppression has been achieved by inhibiting a variety of enzymesincluding for example, the phosphatase calcineurin (inhibited bycyclosporin and FK-506); dihydroorotate dehydrogenase, an enzymeinvolved in the biosynthesis of pyrimidines (inhibited by leflunomideand brequinar); the kinase FRAP (inhibited by rapamycin); and the heatshock protein hsp70 (inhibited by deoxyspergualin). [See B. D. Kahan,Immunological Reviews, 136, pp. 29-49 (1993); R. E. Morris, The Journalof Heart and Lung Transplantation, 12 (6), pp. S275-S286 (1993)].

Inhibitors of IMPDH are also known. U.S. Pat. Nos. 5,380,879 and5,444,072 and PCT publications WO 94/01105 and WO 94/12184 describemycophenolic acid (MPA) and some of its derivatives as potent,uncompetitive, reversible inhibitors of human IMPDH type I (K_(i)=33 nM)and type II (K_(i)=9 nM). MPA has been demonstrated to block theresponse of B and T-cells to mitogen or antigen [A. C. Allison et. al.,Ann. N. Y. Acad. Sci., 696, 63, (1993).

Immunosuppressants, such as MPA, are useful drugs in the treatment oftransplant rejection and autoimmune diseases. [R. E. Morris, KidneyIntl., 49, Suppl. 53, S-26, (1996)]. However, MPA is characterized byundesirable pharmacological properties, such as gastrointestinaltoxicity and poor bioavailability. [L. M. Shaw, et. al., TherapeuticDrug Monitoring, 17, pp. 690-699, (1995)].

Nucleoside analogs such as tiazofurin, ribavirin and mizoribine alsoinhibit IMPDH [L. Hedstrom, et. al. Biochemistry, 29, pp. 849-854(1990)]. These compounds, which are competitive inhibitors of IMPDH,suffer from lack of specificity to this enzyme.

Mycophenolate mofetil, a prodrug which quickly liberates free MPA invivo, was recently approved to prevent acute renal allograft rejectionfollowing kidney transplantation. (L. M. Shaw, et. al., Therapeutic DrugMonitoring, 17, pp. 690-699, (1995); H. W. Sollinger, Transplantation,60, pp. 225-232 (1995)]. Several clinical observations, however, limitthe therapeutic potential of this drug. [L. M. Shaw, et. al.,Therapeutic Drug Monitoring, 17, pp. 690-699, (1995)]. MPA is rapidlymetabolized to the inactive glucuronide in vivo. [A. C., Allison and E.M. Eugui, Immunological Reviews, 136, pp. 5-28 (1993)]. The glucuronidethen undergoes enterohepatic recycling causing accumulation of MPA inthe gastrointestinal tract where it cannot exert its IMPDH inhibitoryactivity on the immune system. This effectively lowers the drug's invivo potency, while increasing its undesirable gastrointestinal sideeffects.

It is also known that IMPDH plays a role in other metabolic events.Increased IMPDH activity has been observed in rapidly proliferatinghuman leukemic cell lines and other tumor cell lines, indicating IMPDHas a target for anti-cancer as well as immunosuppressive chemotherapy[M. Nagai et. al., Cancer Res., 51, pp. 3886-3890, (1991)]. IMPDH hasalso been shown to play a role in the proliferation of smooth musclecells, indicating that inhibitors of IMPDH, such as MPA or rapamycin,may be useful in preventing restenosis or other hyperproliferativevascular diseases [C. R. Gregory et al., Transplantation, 59, pp. 655-61(1995); PCT publication WO 94/12184; and PCT publication WO 94/01105].

Additionally, IMPDH has been shown to play a role in viral replicationin some viral cell lines. [S. F. Carr, J. Biol. Chem., 268, pp.27286-27290 (1993)]. Analogous to lymphocyte and tumor cell lines, theimplication is that the de novo, rather than the salvage, pathway iscritical in the process of viral replication.

The IMPDH inhibitor ribavirin is currently being evaluated for thetreatment of hepatitis C virus (HCV) and hepatitis B virus (HBV)infection and disease. Ribavirin enhances the sustained efficacy ofinterferon in HBV and HCV treatment. However, the therapeutic potentialof ribavirin is limited by its lack of a sustained response inmonotherapy and broad cellular toxicity.

Thus, there remains a need for potent IMPDH inhibitors with improvedpharmacological properties. Such inhibitors would have therapeuticpotential as immunosuppressants, anti-cancer agents, anti-vascularhyperproliferative agents, antiinflammatory agents, antifungal agents,antipsoriatic and anti-viral agents.

SUMMARY OF THE INVENTION

The present invention provides compounds, and pharmaceuticallyacceptable derivatives thereof, that are useful as inhibitors of IMPDH.These compounds can be used alone or in combination with othertherapeutic or prophylactic agents, such as anti-virals,anti-inflammatory agents, antibiotics, and immunosuppressants for thetreatment or prophylaxis of transplant rejection and autoimmune disease.Additionally, these compounds are useful, alone or in combination withother agents, as therapeutic and prophylactic agents for antiviral,anti-tumor, anti-cancer, antiinflammatory agents, antifungal agents,antipsoriatic immunosuppressive chemotherapy and restenosis therapyregimens.

The invention also provides pharmaceutical compositions comprising thecompounds of this invention, as well as multi-component compositionscomprising additional IMPDH compounds together with animmunosuppressant. The invention also provides methods of using thecompounds of this invention, as well as other related compounds, for theinhibition of IMPDH.

The compounds of this invention, as well as those used in the methods ofthis invention demonstrate a different metabolic profile than MPA andits derivatives. Because of this difference, methods of this inventionand the compounds used therein may offer advantages as therapeutics forIMPDH mediated disease. These advantages include increased overalltherapeutic benefit and reduction in deleterious side effects.

DETAILED DESCRIPTION OF THE INVENTION

In order that the invention herein described may be more fullyunderstood, the following detailed description is set forth. In thedescription, the following abbreviations are used:

Designation Reagent or Fragment Ac acetyl Me methyl Et ethyl Bn benzylCDI carbonyldiimidazole DIEA diisopropylethylamine DMAPdimethylaminopyridine DMF dimethylformamide DMSO dimethylsulfoxide EDC1-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride EtOAc ethylacetate THF tetrahydrofuran

The following terms are employed herein:

Unless expressly stated to the contrary, the terms “—SO₂—” and “—S(O)₂—”as used herein refer to a sulfone or sulfone derivative (i.e., bothappended groups linked to the S), and not a sulfinate ester.

The terms “halo” or “halogen” refer to a radical of fluorine, chlorine,bromine or iodine.

The term “immunosuppressant” refers to a compound or drug whichpossesses immune response inhibitory activity. Examples of such agentsinclude cyclosporin A, FK506, rapamycin, leflunomide, deoxyspergualin,prednisone, azathioprine, mycophenolate mofetil, OKT3, ATAG, interferonand mizoribine.

The term “interferon” refers to all forms of interferons, including butnot limited to alpha, beta and gamma forms.

IMPDH-mediated disease refers to any disease state in which the IMPDHenzyme plays a regulatory role in the metabolic pathway of that disease.Examples of IMPDH-mediated disease include transplant rejection andautoimmune diseases, such as rheumatoid arthritis, multiple sclerosis,juvenile diabetes, asthma, and inflammatory bowel disease, as well asinflammatory diseases, cancer, viral replication diseases and vasculardiseases.

For example, the compounds, compositions and methods of using them ofthis invention may be used in the treatment of transplant rejection(e.g., kidney, liver, heart, lung, pancreas (islet cells), bone marrow,cornea, small bowel and skin allografts and heart valve xenografts) andautoimmune diseases, such as rheumatoid arthritis, multiple sclerosis,juvenile diabetes, asthma, inflammatory bowel disease (Crohn's disease,ulcerative colitus), lupus, diabetes, mellitus myasthenia gravis,psoriasis, dermatitis, eczema, seborrhoea, pulmonary inflammation, eyeuveitis, hepatitis, Grave's disease, Hashimoto's thyroiditis, Behcet'sor Sjorgen's syndrome (dry eyes/mouth), pernicious or immunohaemolyticanaemia, idiopathic adrenal insufficiency, polyglandular autoimmunesyndrome, and glomerulonephritis, scleroderma, lichen planus, viteligo(depigmentation of the skin), autoimmune thyroiditis, and alveolitis,inflammatory diseases such as osteoarthritis, acute pancreatitis,chronic pancreatitis, asthma and adult respiratory distress syndrome, aswell as in the treatment of cancer and tumors, such as solid tumors,lymphomas and leukemia, vascular diseases, such as restenosis, stenosisand artherosclerosis, and DNA and RNA viral replication diseases, suchas retroviral diseases, and herpes.

Additionally, IMPDH enzymes are also known to be present in bacteria andthus may regulate bacterial growth. As such, the IMPDH-inhibitorcompounds, compositions and methods described herein may be useful intreatment or prevention of bacterial infection, alone or in combinationwith other antibiotic agents.

The term “treating” as used herein refers to the alleviation of symptomsof a particular disorder in a patient or the improvement of anascertainable measurement associated with a particular disorder. As usedherein, the term “patient” refers to a mammal, including a human.

The term “thiocarbamates” refers to compounds containing the functionalgroup N—SO₂—O.

The terms “HBV”, “HCV” and “HGV” refer to hepatitis-B virus, hepatitis-Cvirus and hepatitis-G virus, respectively.

According to one embodiment, the invention provides methods ofinhibiting IMPDH activity in a mammal comprising the step ofadministering to said mammal, a compound of formula I:

wherein:

A is selected from:

(C₁-C₆)-straight or branched alkyl, or (C₂-C₆)-straight or branchedalkenyl or alkynyl; and A optionally comprises up to 2 substituents,wherein:

the first of said substituents, if present, is selected from R¹ or R³,and

the second of said substituents , if present, is R¹;

B is a saturated, unsaturated or partially saturated monocyclic orbicyclic ring system optionally comprising up to 4 heteroatoms selectedfrom N, O, or S and selected from the formulae:

 wherein each X is the number of hydrogen atoms necessary to completeproper valence;

and B optionally comprises up to 3 substituents, wherein:

the first of said substituents, if present, is selected from R¹, R², R⁴or R⁵,

the second of said substituents, if present, is selected from R¹ or R⁴,and

the third of said substituents, if present, is R¹; and

D is selected from C(O), C(S), or S(O)₂; wherein:

each R¹ is independently selected from 1,2-methylenedioxy,1,2-ethylenedioxy, R⁶ or (CH₂)_(n)—Y;

wherein n is 0, 1 or 2; and

Y is selected from halogen, CN, NO₂, CF₃, OCF₃, OH, SR⁶, S(O)R⁶, SO₂R⁶,NH₂, NHR⁶, N(R⁶)₂, NR⁶R⁸, COOH, COOR⁶ or OR⁶;

each R² is independently selected from (C₁-C₄)-straight or branchedalkyl, or (C₂-C₄)-straight or branched alkenyl or alkynyl; and each R²optionally comprises up to 2 substituents, wherein:

the first of said substituents, if present, is selected from R¹, R⁴ andR⁵, and

the second of said substituents, if present, is R¹;

R³ is selected from a monocyclic or a bicyclic ring system consisting of5 to 6 members per ring, wherein said ring system optionally comprisesup to 4 heteroatoms selected from N, O, or S, and wherein a CH₂ adjacentto any of said N, O, or S heteroatoms is optionally substituted withC(O); and each R³ optionally comprises up to 3 substituents, wherein:

the first of said substituents, if present, is selected from R¹, R², R⁴or R⁵,

the second of said substituents, if present, is selected from R¹ or R⁴,and

the third of said substituents, if present, is R¹;

each R⁴ is independently selected from OR⁵, OC(O)R⁶, OC(O)R⁵, OC(O)OR⁶,OC(O)OR⁵, OC(O)N(R⁶)₂, OP(O)(OR⁶)₂, SR⁶, SR⁵, S(O)R⁶, S(O)R⁵, SO₂R⁶,SO₂R⁵, SO₂N(R⁶)₂, SO₂NR⁵R⁶, SO₃R⁶, C(O)R⁵, C(O)OR⁵, C(O)R⁶, C(O)OR⁶,NC(O)C(O)R⁶, NC(O)C(O)R⁵, NC(O)C(O)OR⁶, NC(O)C(O)N(R⁶)₂, C(O)N(R⁶)₂,C(O)N(OR⁶)R⁶, C(O)N(OR⁶)R⁵, C(NOR⁶)R⁶, C(NOR⁶)R⁵, N(R⁶)₂, NR⁶C(O)R¹,NR⁶C(O)R⁶, NR⁶C(O)R⁵, NR⁶C(O)OR⁶, NR⁶C(O)OR⁵, NR⁶C(O)N(R⁶)₂,NR⁶C(O)NR⁵R⁶, NR⁶SO₂R⁶, NR⁶SO₂R⁵, NR⁶SO₂N(R⁶)₂, NR⁶SO₂NR⁵R⁶, N(OR⁶)R⁶,N(OR⁶)R⁵, P(O)(OR⁶)N(R⁶)₂, and P(O)(OR⁶)₂;

each R⁵ is a monocyclic or a bicyclic ring system consisting of 5 to 6members per ring, wherein said ring system optionally comprises up to 4heteroatoms selected from N, O, or S, and wherein a CH₂ adjacent to saidN, O or S maybe substituted with C(O); and each R⁵ optionally comprisesup to 3 substituents, each of which, if present, is R¹;

each R⁶ is independently selected from H, (C₁-C₄)-straight or branchedalkyl, or (C₂-C₄) straight or branched alkenyl; and each R⁶ optionallycomprises a substituent that is R⁷;

R⁷ is a monocyclic or a bicyclic ring system consisting of 5 to 6members per ring, wherein said ring system optionally comprises up to 4heteroatoms selected from N, O, or S, and wherein a CH₂ adjacent to saidN, O or S maybe substituted with C(O); and each R⁷ optionally comprisesup to 2 substituents independently chosen from H, (C₁-C₄)-straight orbranched alkyl, (C₂-C₄) straight or branched alkenyl,1,2-methylenedioxy, 1,2-ethylenedioxy, or (CH₂)_(n)—Z;

wherein n is 0, 1 or 2; and

 Z is selected from halogen, CN, NO₂, CF₃, OCF₃, OH, S(C₁-C₄)-alkyl,SO(C₁-C₄)-alkyl, SO₂(C₁-C₄)-alkyl, NH₂, NH(C₁-C₄)-alkyl,N((C₁-C₄)-alkyl)₂, N((C₁-C₄)-alkyl)R⁸, COOH, C(O)O(C₁-C₄)-alkyl orO(C₁-C₄)-alkyl; and

R⁸ is an amino protecting group; and

wherein any carbon atom in any A, R² or R⁶ is optionally replaced by O,S, SO, SO₂, NH, or N(C₁-C₄)-alkyl.

The term “substituted” refers to the replacement of one or more hydrogenradicals in a given structure with a radical selected from a specifiedgroup. When more than one hydrogen radical may be replaced with asubstituent selected from the same specified group, the substituents maybe either the same or different at every position.

The term “monocyclic or bicyclic ring system consisting of 5 to 6members per ring” refers to 5 or 6 member monocyclic rings and 8, 9 and10 membered bicyclic ring structures, wherein each bond in each ring maybe possess any degree of saturation that is chemically feasible. Whensuch structures contain substituents, those substituents may be at anyposition of the ring system, unless otherwise specified.

As specified, such ring systems may optionally comprise up to 4heteroatoms selected from N, O or S. Those heteroatoms may replace anycarbon atoms in these ring systems as long as the resulting compound ischemically stable.

The term “wherein each X is the number of hydrogen atoms necessary tocomplete proper valence” means that X is 0, 1 or 2 hydrogen atoms,depending upon the identity of the ring atom to which X is bound (C, N,O S), the identity of the two adjacent ring atoms, and the nature of thebonds between the ring atom to which X is bound and the two adjacentring atoms (single, double or triple bond). In essence, this definitionis meant to exclude from X any substituents other than hydrogen.

The term “amino protecting group” refers to a suitable chemical groupwhich may be attached to a nitrogen atom. The term “protected” refers towhen the designated functional group is attached to a suitable chemicalgroup (protecting group). Examples of suitable amino protecting groupsand protecting groups are described in T. W. Greene and P. G. M. Wuts,Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons(1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents forOrganic Synthesis, John Wiley and Sons (1994); L. Paquette, ed.Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons(1995) and are exemplified in certain of the specific compounds used inthis invention.

According to another embodiment, the invention provides methods ofinhibiting IMPDH in mammals by administering a compound of the formula(II):

wherein B and D are as defined above.

More preferably, in methods employing the compounds of formulae (I) or(II), component B comprises from 0 to 2 substituents. According to analternate embodiment, the invention provides methods for inhibitingIMPDH in a mammal employing compounds (I) or (II), wherein B comprisesat least a single substituent selected from the group defined by R⁵.Preferably, in this embodiment, B is a monocyclic aromatic ringcontaining at least one substituent which is also a monocyclic aromaticring.

The present invention also provides compounds which are useful ininhibiting IMPDH. According to one embodiment, the IMPDH inhibitorycompound has the formula (III):

wherein A, B and D are as defined above;

E is oxygen or sulfur; and

G and G′ are independently selected from R¹ or hydrogen.

According to an alternate embodiment, the invention provides a compoundof the formula (IV):

wherein B, D, E, G and G′ are defined as above and B′ is a saturated,unsaturated or partially saturated monocyclic or bicyclic ring systemoptionally comprising up to 4 heteroatoms selected from N, O, or S andselected from the formulae:

wherein each X is the number of hydrogen atoms necessary to completeproper valence;

and B′ optionally comprises up to 3 substituents, wherein:

the first of said substituents, if present, is selected from R¹, R², R⁴or R⁵,

the second of said substituents, if present, is selected from R¹ or R⁴,and

the third of said substituents, if present, is R¹; wherein X, R¹, R², R⁴and R⁵ are defined as above.

Excluded from this invention are compounds of formula (IV) wherein B andB′ are simultaneously unsubstituted phenyl and compounds wherein B isunsubstituted phenyl and B′ is tri-chloro-, tri-bromo or tri-iodophenyl.

Preferably, in compounds of formula (IV), B and B′ are phenyl groupscomprising at least one substituent each. These compounds arerepresented by formula (V):

wherein K is selected from R¹ or R⁴; and J is selected from R¹, R² orR⁴.

Preferred compounds of formula (V) are those wherein D is —C(O)—, thosewherein E is oxygen; those wherein J is NR⁶C(O)R⁵ or NR⁶C(O)R⁶,preferably NR⁶C(O)R⁶, more preferably N(CH₃)C(O)R⁶, and more preferablyN(CH₃)C(O)CH₃; those wherein K is (CH₂)_(n)—Y, preferably OCH₃ (i.e., nis 0, Y is OR⁶, and R⁶ is CH₃); and those wherein G is hydrogen. Morepreferred compounds of formula (V) are those wherein:

E is oxygen

J is NR⁶C(O)R⁵ or NR⁶C(O)R⁶;

K is (CH₂)_(n)—Y; and

G is hydrogen.

Even more preferred compounds of formula (V) are those wherein:

D is —C(O)—;

E is oxygen;

J is NR₆C(O)R⁶;

K is OCH₃; and

G is hydrogen.

Most preferably in such compounds, J is N(CH₃)C(O)R⁶.

Alternate preferred compounds are those of formula V: wherein J is R²,those wherein D is —C(O)—, those wherein E is oxygen, those wherein J isR² substituted with R⁴, preferably wherein R⁴ is NR⁶C(O)OR⁵ orNR⁶C(O)OR⁶, more preferably wherein R⁴ is NR⁶C(O)OR⁵, more preferablywherein R⁴ is NHC(O)OR⁵, and more preferably wherein R⁴ isNHC(O)O—3-tetrahydrofuranyl, those wherein K is (CH₂)_(n)—Y, preferablywherein K is OCH₃, those wherein G is hydrogen, and those wherein:

D is —C(O)—;

E is oxygen;

K is OCH₃; and

G is hydrogen.

Alternatively, other preferred compounds include those of formula VI:

those compounds of formula VI wherein K is OCH₃, and those compounds offormula VI wherein G is hydrogen.

An alternate embodiment of this invention is compounds of formula Vwherein K is selected from R¹ or R⁴; and J is selected from R¹, R², R⁴,and R⁹ wherein, R¹, R², and R⁴, are as defined above and R⁹ isindependently selected from (C₁-C₄)-straight or branched alkyl, or(C₂-C₄)-straight or branched alkenyl or alkynyl; and each R⁹ optionallycomprises up to 2 substituents selected from NR⁶C(O)OR¹⁰, wherein R6 isas defined above and R¹⁰ is selected from (C₁-C₅)-straight or branchedalkyl optionally comprising up to two substituents selected from NR⁶R⁸,SR⁶, SO₂R⁶, —(CH₂)_(n)—SR⁶, —(CH₂)_(n)—OR⁶, and OR⁶, wherein n, R⁶ andR⁸, are as defined above.

In another embodiment, preferred compounds are those of formula VII:

wherein K is selected from R¹ and R⁴; and

A, D, R¹ and R⁴ are each independently as defined in claim 1.

More preferred compounds of formula VII are those wherein D is —C(O)—,those wherein A is a monocyclic aromatic ring substituted with 1-2substituents selected from the group consisting of NR⁶C(O)R⁶, NR⁶C(O)R⁵,CH₂NR⁶C(O)OR⁶, and CH₂NR⁶C(O)OR⁵, those wherein A is a monocyclicaromatic ring substituted with 1-2 substituents selected from the groupconsisting of CH₂NR⁶C(O)OR⁶ and CH₂NR⁶C(O)OR⁵, those A is a monocyclicaromatic ring substituted with CH₂NR⁶C(O)OR⁵, those wherein A is amonocyclic aromatic ring substituted with CH₂NHC(O)OR⁵, those wherein Ais a monocyclic aromatic ring substituted withCH₂NHC(O)O-3-tetrahydrofuryl, those wherein K is (CH₂)_(n)—Y, thosewherein K is OCH₃, and those wherein:

A is a monocyclic aromatic ring substituted withCH₂NHC(O)O-3-tetrahydrofuryl; and

K is OCH₃.

Alternatively, other preferred compounds of this invention include thosecompounds of formula VIII:

wherein D and K are as defined in claim 1.

Another embodiment is those compounds of formula IX:

wherein:

D is selected from C(O), C(S) and S(O)₂;

K is selected from R¹ and R⁴; and

J is selected from R¹, R², and R⁴.

More preferred compounds of formula IX include those wherein D is—C(O)—, those wherein J is NR⁶C(O)R⁵ or NR⁶C(O)R⁶, those wherein J isNR⁶C(O)R⁶, those wherein J is N(CH₃)C(O)R⁶, those wherein J isN(CH₃)C(O)CH₃, those wherein K is (CH₂)_(n)—Y, those wherein K is OCH₃,and those wherein:

K is OCH₃; and

J is N(CH₃)C(O)CH₃.

Tables IA, IB and IIB list preferred individual compounds of theinvention and preferred compounds employed in the compositions andmethods of this invention. Table IIA lists preferred compounds employedin the methods of this invention.

TABLE IA

# G K A 1 H H benzyl

TABLE IB

# G K B^(′) 2 H H 3-methoxyphenyl 3 H H 3-thienyl 4 H H3,4-difluorophenyl 5 H H 2,5-dimethoxyphenyl 6 H H 3-methylthiophenyl 7H H 3-bromophenyl 8 H H 3-cyanophenyl 9 H H 3-trifluoromethyl-4-chlorophenyl 10 H H 2-methyl-3-chlorophenyl 11 H H2-methoxy-5-methylphenyl 12 H H 2-methoxyphenyl 13 H H 3-methoxyphenyl14 H H 2,5-dimethoxyphenyl 15 H H 3-nitrophenyl 16 H H 4-nitrophenyl 17H H 3-methylphenyl 18 H H 3-trifluoromethylphenyl 19 H H2-trifluoromethylphenyl 20 H H 3-fluorophenyl 21 H H 4-phenoxyphenyl 22H H 3-chlorophenyl 23 H H 3-chloro-4-fluorophenyl 24 H H 3-aminophenyl25 H H 3-(hydroxymethyl)phenyl 26 H H 3-acetylenylphenyl 27 H H3-hydroxyphenyl 29 H H 3-pyridinyl 30 H H 4-pyridinyl 31 H H2-(5-methyl)thiazolyl 39 H H 3,4-ethylenedioxyphenyl 40 H H3-methyl-4-nitrophenyl 41 H H 3-trifluoromethyl-4- nitrophenyl 42 H3-chloro phenyl 43 H 3-chloro 3-methylphenyl 44 — — — 45 H 3-fluorophenyl 46 H 3-fluoro 3-methylphenyl 47 H H 3-carbomethoxymethylphenyl 48H H 3-carboxyethylphenyl 49 H H 3-dimethylaminophenyl 50 H H 3-[2-(2-methyl)dioxolanyl]phenyl 51 H H 3-aminocarbonylphenyl 53 H H3-(3-furanyl)-phenyl 54 H H 3-carboxymethylphenyl 55 H 3-methoxy3-methylphenyl 56 H 3-methoxy 3-nitrophenyl 57 H 3-chloro3-carbomethoxymethylphenyl 58 H H 3-amino-5-methylphenyl 59 H 3-methoxy3-aminophenyl 60 H 3-bromo 3-methylphenyl 61 H 3-chloro 3-chloro-4-(5-oxazolyl)phenyl 62 H 3-chloro 4-(2-methylpyridyl) 63 H 3-chloro3-carboxymethylphenyl 64 H 3-bromo 3-nitrophenyl 65 H 3-bromo3-aminophenyl 66 H H 3-[5-(2- methylpyrimidinyl)]phenyl 67 H H3-(5-oxazolyl)phenyl 68 H 3-chloro 2-thienyl 69 H 3-chloro 3-thienyl 71H 3-chloro 3-methoxycarbamoyl-phenyl 72 H 3-chloro 3-acetamidophenyl 73H 3-chloro 3-iodophenyl 74 H 3-methyl phenyl 75 H 3-methyl3-methylphenyl 76 methyl 3-chloro 3-methylphenyl 77 methyl H3-methylphenyl 78 H 3-chloro 3-nitrophenyl 79 H 3-chloro 3-aminophenyl80 H H 3- (cyclohexylsulfamoyl)phenyl 81 H H 3-(methylsufamoyl)phenyl 82H H 3-(phenylsufamoyl)phenyl 83 H 3-methoxy 3-benzyloxycarbamoyl-phenyl84 H 3-methoxy 3-acetamidophenyl 85 H 3-chloro 4-(2-methyl)furanyl 86 H3-chloro 5-(2-methyl)thienyl 88 H 3-carbomethoxy 3-methylphenyl 89 H3-carbomethoxy 3-nitrophenyl 91 H 3-chloro 4-(2-nitro)thienyl 92 H3-chloro 4-(2-hydroxyamino)thienyl 93 H 3-chloro 3-(N-methyl)trifluoroacetamido- phenyl 94 H 3-chloro 3-(methylamino)phenyl 95H 3-chloro 4-(2-amino)thienyl 96 H 3-methoxy 3-trifluoroacetamidophenyl97 H 3-methoxy 3-(N- methyl)trifluoroacetamido- phenyl 98 H 3-methoxy3-)3′- picolyloxycarbamoyl)phenyl 99 H 3-methoxy3-(phenoxycarbamoyl)phenyl 100 H 3-methoxy 3-difluoroacetamidophenyl 101H 3- 3-methylphenyl acetoxymethyl 102 H 3- 3-methylphenyl hydroxymethyl104 H H 3-nitro-4-fluorophenyl 105 H 3-methoxy 3-(aminomethyl)phenyl[*TFA] 106 H 3-methoxy 5-(N-acetoxy)indolinyl 107 H 3-methoxy3-(N-methyl)acetamidophenyl 108 H 3-methoxy 3-[(2-oxo-2-(3,4,5-tri-methoxyphenyl)acetyl) amino]phenyl 109 H 3-amino 3-methylphenyl 110 H3-methoxy 3-benzamidophenyl 111 H 3-methoxy 3-phenylacetamidophenyl 112H 3-methoxy 3-phenylureidophenyl 113 H 3-methoxy 3-(t-butoxycarbamoylmethyl)phenyl 114 H 3-methoxy 3-(cyclopentylacetamido) phenyl 115 H3-methoxy 3-methylphenyl

TABLE IC

Compound L 116 NHC(O)O-t-butyl 117 NCH₃C(O)O-t-butyl 118 NHC(O)O-methyl119 NHC(O)O-phenyl 120 NHC(O)O-(S)-3-tetrahydrofuranyl 121NHC(O)O-2-picolinyl 122 NHC(O)O-(S)-5-oxazolidinonylmethyl 123NHC(O)O-4-carbomethoxyphenyl 124 NHC(O)O-isobutyl 125 NHC(O)O-allyl 126NHC(O)O-5-(1,3-dioxanyl) 127 NHC(O)O-4-acetamidophenyl 128NHC(O)O-2-furfuryl 129 NHC(O)O-2-thiofurfuryl 130 NHC(O)O-2-methoxyethyl131 NHC(O)O-4-tetrahydropyranyl 132 NHC(O)O-cyclohexyl 133NHC(O)O-cyclopentyl 134 NHC(O)O-2-hydroxyethyl 135NHC(O)O-cyclohexylmethyl 136 NHC(O)O-(R,S)-3-tetrahydrofuranyl 137NHC(O)O-3-pyridyl 138 NHC(O)O-benzyl 139 NHC(O)O-3-(tBOC-amino)propyl140 NHC(O)O-4-hydroxybutyl 141 NHC(O)O-5-hydroxypentyl 142NHC(O)O-(R,S)-2-pyranyl 143 NHC(O)O-3-(N-tBOC)-piperidinyl 144NHC(O)O-(R)-3-(2-oxo-4,4- dimethyl)furanyl 145NHC(O)O-3-methylthiopropyl 146 NHC(O)O-4-[(2,2-dimethyl)-1,3-dioxanyl]methyl 147 NHC(O)O-2-di-(hydroxymethyl)ethyl 148NHC(O)O-4-(N-tBOC)-piperidinylmethyl 149NHC(O)O-3-(N-tBOC)-piperidinylmethyl 150NHC(O)O-(dibenzyloxymethyl)methyl 151 NHC(O)O-di-(hydroxymethyl)methyl152 NHC(O)O-2-(N-tBOC)-piperidinylmethyl 153 NHC(O)O-3-piperidinyl-TFA154 NHC(O)O-(R,S)-(2- tetrahydropyranyl) methyl 155NHC(O)O-4-piperidinylmethyl-TFA 156NHC(O)O-(R,S)-tetrahydrofuranylmethyl 157 NHC(O)O-3-methylsulfonylpropyl158 NHC(O)O-3-piperidinylmethyl-TFA 159 NHC(O)O-2-piperidinylmethyl-TFA160 NHC(O)O-(R,S)-3-tetrahydrothiophenyl 161NHC(O)O-(R,S)-3-tetrahydrothiopyranyl 162 NHC(O)O-3-methoxypropyl

TABLE IIA

# Q¹ Q² B 28 3-methoxy 4-methoxy 3-methylphenyl 32 3-nitro H3-methylphenyl 33 4-cyano H 3-methylphenyl 34 3-methoxy 4-methoxy3-bromophenyl 35 3-methoxy 4-methoxy 2-methoxy-5- chlorophenyl 363-methoxy 4-methoxy 3-fluorophenyl 37 3-methoxy 4-methoxy 3-ethylphenyl38 3-methoxy 4-methoxy 3-methylthiophenyl 52 3-chloro 4-methoxy3-nitrophenyl 70 4-cyano 3-chloro 3-methylphenyl 87 1-imidazolyl H3-methylphenyl 90 3-hydroxymethyl 4-methoxy 3-methylphenyl 1033-(t-butoxycarbamoyl H 3-(t-butoxycarbamoyl methyl) methyl)phenyl

TABLE IIB

# Q₁ Q₃ 163 Cl N(Me) (Ac) 164 OMe N(Me) (Ac) 165 SMe CH₂NHC(O)O-(3s)-tetrahydrofuranyl 166 S(O)₂Me N(Me) (Ac) 167 OMe N(Me) (Ac) 168 SMeCH₂NHC(O)O-(3s)- tetrahydrofuranyl

The compounds of Table IIA correspond to compounds of formula (II)wherein one of said B components is phenyl with two substituents, Q¹ andQ². In accordance with formula (II):

Q¹ is selected from R¹, R², R⁴ or R⁵; and

Q² is selected from R¹ or R⁴.

The compounds of this invention may contain one or more asymmetriccarbon atoms and thus may occur as racemates and racemic mixtures,single enantiomers, diastereomeric mixtures and individualdiastereomers. All such isomeric forms of these compounds are expresslyincluded in the present invention. Each stereogenic carbon may be of theR or S configuration.

Combinations of substituents and variables envisioned by this inventionare only those that result in the formation of stable compounds. Theterm “stable”, as used herein, refers to compounds which possessstability sufficient to allow manufacture and which maintains theintegrity of the compound for a sufficient period of time to be usefulfor the purposes detailed herein (e.g., therapeutic or prophylacticadministration to a mammal or for use in affinity chromatographyapplications). Typically, such compounds are stable at a temperature of40° C. or less, in the absence of moisture or other chemically reactiveconditions, for at least a week.

As used herein, the compounds of this invention, including the compoundsof formulae I-IX, are defined to include pharmaceutically acceptablederivatives or prodrugs thereof. A “pharmaceutically acceptablederivative or prodrug” means any pharmaceutically acceptable salt,ester, salt of an ester, or other derivative of a compound of thisinvention which, upon administration to a recipient, is capable ofproviding (directly or indirectly) a compound of this invention.Particularly favored derivatives and prodrugs are those that increasethe bioavailability of the compounds of this invention when suchcompounds are administered to a mammal (e.g., by allowing an orallyadministered compound to be more readily absorbed into the blood) orwhich enhance delivery of the parent compound to a biologicalcompartment (e.g., the brain or lymphatic system) relative to the parentspecies. Preferred prodrugs include derivatives where a group whichenhances aqueous solubility or active transport through the gut membraneis appended to the structure of formulae I-IX.

Pharmaceutically acceptable salts of the compounds of this inventioninclude those derived from pharmaceutically acceptable inorganic andorganic acids and bases. Examples of suitable acid salts includeacetate, adipate, alginate, aspartate, benzoate, benzenesulfonate,bisulfate, butyrate, citrate, camphorate, camphorsulfonate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptanoate, glycerophosphate, glycolate,hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate,palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, salicylate, succinate, sulfate, tartrate,thiocyanate, tosylate and undecanoate. Other acids, such as oxalic,while not in themselves pharmaceutically acceptable, may be employed inthe preparation of salts useful as intermediates in obtaining thecompounds of the invention and their pharmaceutically acceptable acidaddition salts.

Salts derived from appropriate bases include alkali metal (e.g.,sodium), alkaline earth metal (e.g., magnesium), ammonium and N-(C₁₋₄alkyl)₄ ⁺ salts. This invention also envisions the quaternization of anybasic nitrogen-containing groups of the compounds disclosed herein.Water or oil-soluble or dispersible products may be obtained by suchquaternization.

The compounds of this invention may be synthesized using conventionaltechniques. Advantageously, these compounds are conveniently synthesizedfrom readily available starting materials.

In general, compounds of formula (I)-(IX) are conveniently obtained viamethods illustrated in General Synthetic Schemes 1-3.

In General Synthetic Scheme 1 (see below), an X-substituted aniline isreacted with a Y-substituted phenylisocyanate under standard conditionsto give the desired urea. In this process, X and Y may be one or moreindependent substituents (or their suitably protected variants) asexemplified by the ring substituents listed for compounds of formulaeI-IX above, at any position on the aromatic ring.

In General Synthetic Scheme 2 (see above), a substituted benzaldehyde(here, 2-methoxy-4-nitro-substituted) is treated sequentially withtosylmethylisocyanide, to give the resulting oxazole, then reduced bycatalytic hydrogenation to give the desired aniline. Reaction of thisaniline with an isocyanate (here, m-tolylisocyanate) under standardconditions gives the desired urea.

An alternate synthetic route is illustrated in General Synthetic Scheme3 (see above). A substituted benzaldehyde (here 4-nitro substituted) isconverted to the corresponding oxazolyl aniline as shown in GeneralSynthetic Scheme 2. This aniline is treated with a substituted benzoicacid (here, 3-methyl-substituted) and a carboxylic acid activatingagent, such as diphenylphosphoryl azide, under standard reactionconditions, to give the desired urea.

As can be appreciated by the skilled artisan, the above syntheticschemes are not intended to comprise a comprehensive list of all meansby which the compounds described and claimed in this application may besynthesized. Further methods will be evident to those of ordinary skillin the art. Additionally, the various synthetic steps described abovemay be performed in an alternate sequence or order to give the desiredcompounds.

The compounds of this invention may be modified by appending appropriatefunctionalities to enhance selective biological properties. Suchmodifications are known in the art and include those which increasebiological penetration into a given biological compartment (e.g., blood,lymphatic system, central nervous system), increase oral availability,increase solubility to allow administration by injection, altermetabolism and alter rate of excretion.

The novel compounds of the present invention are excellent ligands forIMPDH. Accordingly, these compounds are capable of targeting andinhibiting IMPDH enzyme. Inhibition can be measured by various methods,including, for example, IMP dehydrogenase HPLC assays (measuringenzymatic production of XMP and NADH from IMP and NAD) and IMPdehydrogenase spectrophotometric assays (measuring enzymatic productionof NADH from NAD). [See C. Montero et al., Clinica Chimica Acta, 238,pp. 169-178 (1995)].

Pharmaceutical compositions of this invention comprise a compound offormulae (I), (II) or (VII) or a pharmaceutically acceptable saltthereof; an additional agent selected from an immunosuppressant, ananti-cancer agent, an anti-viral agent, antiinflammatory agent,antifungal agent, antibiotic, or an anti-vascular hyperproliferationcompound; and any pharmaceutically acceptable carrier, adjuvant orvehicle. Alternate compositions of this invention comprise a compound offormulae (III)-(IX) or a pharmaceutically acceptable salt thereof; and apharmaceutically acceptable carrier, adjuvant or vehicle. Suchcomposition may optionally comprise an additional agent selected from animmunosuppressant, an anti-cancer agent, an anti-viral agent,antiinflammatory agent, antifungal agent, antibiotic, or ananti-vascular hyperproliferation compound.

The term “pharmaceutically acceptable carrier or adjuvant” refers to acarrier or adjuvant that may be administered to a patient, together witha compound of this invention, and which does not destroy thepharmacological activity thereof and is nontoxic when administered indoses sufficient to deliver a therapeutic amount of the compound.

Pharmaceutically acceptable carriers, adjuvants and vehicles that may beused in the pharmaceutical compositions of this invention include, butare not limited to, ion exchangers, alumina, aluminum stearate,lecithin, self-emulsifying drug delivery systems (SEDDS) such asdα-tocopherol polyethyleneglycol 1000 succinate, surfactants used inpharmaceutical dosage forms such as Tweens or other similar polymericdelivery matrices, serum proteins, such as human serum albumin, buffersubstances such as phosphates, glycine, sorbic acid, potassium sorbate,partial glyceride mixtures of saturated vegetable fatty acids, water,salts or electrolytes, such as protamine sulfate, disodium hydrogenphosphate, potassium hydrogen phosphate, sodium chloride, zinc salts,colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat. Cyclodextrins such as α-, β-, and γ-cyclodextrin, orchemically modified derivatives such as hydroxyalkylcyclodextrins,including 2- and 3-hydroxypropyl-β-cyclodextrins, or other solubilizedderivatives may also be advantageously used to enhance delivery ofcompounds of formulae I-IX.

The pharmaceutical compositions of this invention may be administeredorally, parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. We prefer oraladministration or administration by injection. The pharmaceuticalcompositions of this invention may contain any conventional non-toxicpharmaceutically-acceptable carriers, adjuvants or vehicles. In somecases, the pH of the formulation may be adjusted with pharmaceuticallyacceptable acids, bases or buffers to enhance the stability of theformulated compound or its delivery form. The term parenteral as usedherein includes subcutaneous, intracutaneous, intravenous,intramuscular, intra-articular, intraarterial, intrasynovial,intrasternal, intrathecal, intralesional and intracranial injection orinfusion techniques.

The pharmaceutical compositions may be in the form of a sterileinjectable preparation, for example, as a sterile injectable aqueous oroleaginous suspension. This suspension may be formulated according totechniques known in the art using suitable dispersing or wetting agents(such as, for example, Tween 80) and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example, as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are mannitol, water, Ringer'ssolution and isotonic sodium chloride solution. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium. For this purpose, any bland fixed oil may be employed includingsynthetic mono- or diglycerides. Fatty acids, such as oleic acid and itsglyceride derivatives are useful in the preparation of injectables, asare natural pharmaceutically-acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oilsolutions or suspensions may also contain a long-chain alcohol diluentor dispersant such as those described in Pharmacopeia Helvetica, Ph.Helv., or a similar alcohol, or carboxymethyl celluose or similardispersing agents which are commonly used in the formulation ofpharmaceutically acceptable dosage forms such as emulsions and orsuspensions Other commonly used surfactants such as Tweens or Spansand/or other similar emulsifying agents or bioavailability enhancerswhich are commonly used in the manufacture of pharmaceuticallyacceptable solid, liquid, or other dosage forms may also be used for thepurposes of formulation.

The pharmaceutical compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, emulsions and aqueous suspensions,dispersions and solutions. In the case of tablets for oral use, carrierswhich are commonly used include lactose and corn starch. Lubricatingagents, such as magnesium stearate, are also typically added. For oraladministration in a capsule form, useful diluents include lactose anddried corn starch. When aqueous suspensions and/or emulsions areadministered orally, the active ingredient may be suspended or dissolvedin an oily phase is combined with emulsifying and/or suspending agents.If desired, certain sweetening and/or flavoring and/or coloring agentsmay be added.

The pharmaceutical compositions of this invention may also beadministered in the form of suppositories for rectal administration.These compositions can be prepared by mixing a compound of thisinvention with a suitable non-irritating excipient which is solid atroom temperature but liquid at the rectal temperature and therefore willmelt in the rectum to release the active components. Such materialsinclude, but are not limited to, cocoa butter, beeswax and polyethyleneglycols.

Topical administration of the pharmaceutical compositions of thisinvention is especially useful when the desired treatment involves areasor organs readily accessible by topical application. For applicationtopically to the skin, the pharmaceutical composition should beformulated with a suitable ointment containing the active componentssuspended or dissolved in a carrier. Carriers for topical administrationof the compounds of this invention include, but are not limited to,mineral oil, liquid petroleum, white petroleum, propylene glycol,polyoxy-ethylene polyoxypropylene compound, emulsifying wax and water.Alternatively, the pharmaceutical composition can be formulated with asuitable lotion or cream containing the active compound suspended ordissolved in a carrier with suitable emulsifying agents. Suitablecarriers include, but are not limited to, mineral oil, sorbitanmonostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol,2-octyldodecanol, benzyl alcohol and water. The pharmaceuticalcompositions of this invention may also be topically applied to thelower intestinal tract by rectal suppository formulation or in asuitable enema formulation. Topically-transdermal patches are alsoincluded in this invention.

The pharmaceutical compositions of this invention may be administered bynasal aerosol or inhalation. Such compositions are prepared according totechniques well-known in the art of pharmaceutical formulation and maybe prepared as solutions in saline, employing benzyl alcohol or othersuitable preservatives, absorption promoters to enhance bioavailability,fluorocarbons, and/or other solubilizing or dispersing agents known inthe art.

Dosage levels of between about 0.01 and about 100 mg/kg body weight perday, preferably between about 0.5 and about 75 mg/kg body weight per dayof the IMPDH inhibitory compounds described herein are useful in amonotherapy and/or in combination therapy for the prevention andtreatment of IMPDH mediated disease. Typically, the pharmaceuticalcompositions of this invention will be administered from about 1 toabout 5 times per day or alternatively, as a continuous infusion. Suchadministration can be used as a chronic or acute therapy. The amount ofactive ingredient that may be combined with the carrier materials toproduce a single dosage form will vary depending upon the host treatedand the particular mode of administration. A typical preparation willcontain from about 5% to about 95% active compound (w/w). Preferably,such preparations contain from about 20% to about 80% active compound.

When the compositions of this invention comprise a combination of anIMPDH inhibitor of formulae (I)-(IX) and one or more additionaltherapeutic or prophylactic agents, both the IMPDH inhibitor and theadditional agent should be present at dosage levels of between about 10to 100%, and more preferably between about 10 to 80% of the dosagenormally administered in a monotherapy regimen. The additional agentsmay be administered separately, as part of a multiple dose regimen, fromthe compounds of this invention. Alternatively, those agents may be partof a single dosage form, mixed together with the compounds of thisinvention in a single composition.

According to one embodiment, the pharmaceutical compositions of thisinvention comprise an additional immunosuppression agent. Examples ofadditional immunosuppression agents include, but are not limited to,cyclosporin A, FK506, rapamycin, leflunomide, deoxyspergualin,prednisone, azathioprine, mycophenolate mofetil, OKT3, ATAG, interferonand mizoribine.

According to an alternate embodiment, the pharmaceutical compositions ofthis invention may additionally comprise an anti-cancer agent. Examplesof anti-cancer agents include, but are not limited to, cis-platin,actinomycin D, doxorubicin, vincristine, vinblastine, etoposide,amsacrine, mitoxantrone, tenipaside, taxol, colchicine, cyclosporin A,phenothiazines, interferon and thioxantheres.

According to another alternate embodiment, the pharmaceuticalcompositions of this invention may additionally comprise an anti-viralagent. Examples of anti-viral agents include, but are not limited to,Cytovene, Ganciclovir, trisodium phosphonoformate, Ribavirin, d4T, ddI,AZT, and acyclovir.

According to yet another alternate embodiment, the pharmaceuticalcompositions of this invention may additionally comprise ananti-vascular hyperproliferative agent. Examples of anti-vascularhyperproliferative agents include, but are not limited to, HMG Co-Areductase inhibitors such as lovastatin, thromboxane A2 synthetaseinhibitors, eicosapentanoic acid, ciprostene, trapidil, ACE inhibitors,low molecular weight heparin, mycophenolic acid, rapamycin and5-(3′-pyridinylmethyl)benzofuran-2-carboxylate.

Upon improvement of a patient's condition, a maintenance dose of acompound, composition or combination of this invention may beadministered, if necessary. Subsequently, the dosage or frequency ofadministration, or both, may be reduced, as a function of the symptoms,to a level at which the improved condition is retained when the symptomshave been alleviated to the desired level, treatment should cease.Patients may, however, require intermittent treatment on a long-termbasis upon any recurrence of disease symptoms.

As the skilled artisan will appreciate, lower or higher doses than thoserecited above may be required. Specific dosage and treatment regimensfor any particular patient will depend upon a variety of factors,including the activity of the specific compound employed, the age, bodyweight, general health status, sex, diet, time of administration, rateof excretion, drug combination, the severity and course of theinfection, the patient's disposition to the infection and the judgmentof the treating physician.

In an alternate embodiment, this invention provides methods of treatingor preventing IMPDH mediated disease in a a mammal comprising the stepof administrating to said mammal any of the pharmaceutical compositionsand combinations described above. If the pharmaceutical composition onlycomprises the IMPDH inhibitor of this invention as the active component,such methods may additionally comprise the step of administering to saidmammal an agent selected from an antiinflammatory agent,immunosuppressant, an anti-cancer agent, an anti-viral agent, or ananti-vascular hyperproliferation compound. Such additional agent may beadministered to the mammal prior to, concurrently with, or following theadministration of the IMPDH inhibitor composition.

In a preferred embodiment, these methods are useful in suppressing animmune response in a mammal. Such methods are useful in treating orpreventing diseases, including, transplant rejection (e.g., kidney,liver, heart, lung, pancreas (islet cells), bone marrow, cornea, smallbowel and skin allografts and heart valve xenografts), graft versus hostdisease, and autoimmune diseases, such as rheumatoid arthritis, multiplesclerosis, juvenile diabetes, asthma, inflammatory bowel disease(Crohn's disease, ulcerative colitus), lupus, diabetes, mellitusmyasthenia gravis, psoriasis, dermatitis, eczema, seborrhoea, pulmonaryinflammation, eye uveitis, hepatitis, Grave's disease, Hashimoto'sthyroiditis, Behcet's or Sjorgen's syndrome (dry eyes/mouth), perniciousor immunohaemolytic anaemia, idiopathic adrenal insufficiency,polyglandular autoimmune syndrome, glomerulonephritis, scleroderma,lichen planus, viteligo (depigmentation of the skin), autoimmunethyroiditis, and alveolitis.

These methods comprise the step of administering to the mammal acomposition comprising a compound of any of formulae I-IX and apharmaceutically acceptable adjuvant. In a preferred embodiment, thisparticular method comprises the additional step of administering to saidmammal a composition comprising an additional immunosuppressant and apharmaceutically acceptable adjuvant.

Alternatively, this method comprises the step of administering to saidmammal a composition comprising a compound of formulae I-IX; anadditional immunosuppressive agent and a pharmaceutically acceptableadjuvant.

In an alternate preferred embodiment, these methods are useful forinhibiting viral replication in a mammal. Such methods are useful intreating or preventing, DNA and RNA viral diseases caused by, forexample, HTLV-1 and HTLV-2, HIV-1 and HIV-2, nasopharyngeal carcinomavirus, HBV, HCV, HGV, yellow fever virus, dengue fever virus, Japaneseencephalitis virus, human papilloma virus, rhinoviruses and Herpesviruses, such as Epstein-Barr, cytomegaloviruses and Herpes Simplex,Types 1 and 2, or Type 6. [See, U.S. Pat. No. 5,380,879].

These methods comprise the step of administering to the mammal acomposition comprising a compound of any of formulae I-IX, and apharmaceutically acceptable adjuvant. In a preferred embodiment, thisparticular method comprises the additional step of administering to saidmammal a composition comprising an additional anti-viral agent and apharmaceutically acceptable adjuvant.

Alternatively, this method comprises the step of administering to saidmammal a composition comprising a compound of formulae I-IX; anadditional anti-viral agent and a pharmaceutically acceptable adjuvant.

In another alternate preferred embodiment, these methods are useful forinhibiting vascular cellular hyperproliferation in a mammal. Suchmethods are useful in treating or preventing diseases, including,restenosis, stenosis, artherosclerosis and other hyperproliferativevascular disease.

These methods comprise the step of administering to the mammal acomposition comprising a compound of any of formulae I-IX, and apharmaceutically acceptable adjuvant. In a preferred embodiment, thisparticular method comprises the additional step of administering to saidmammal a composition comprising an additional anti-vascularhyperproliferative agent and a pharmaceutically acceptable adjuvant.

Alternatively, this method comprises the step of administering to saidmammal a composition comprising a compound of formulae I-IX; anadditional anti-vascular hyperproliferative agent and a pharmaceuticallyacceptable adjuvant.

In another alternate preferred embodiment, these methods are useful forinhibiting tumors and cancer in a mammal. Such methods are useful intreating or preventing diseases, including, tumors and malignancies,such as lymphoma, leukemia and other forms of cancer.

These methods comprise the step of administering to the mammal acomposition comprising a compound of any of formulae I-IX, and apharmaceutically acceptable adjuvant. In a preferred embodiment, thisparticular method comprises the additional step of administering to saidmammal a composition comprising an additional anti-tumor or anti-canceragent and a pharmaceutically acceptable adjuvant.

Alternatively, this method comprises the step of administering to saidmammal a composition comprising a compound of formulae I-IX; anadditional anti-tumor or anti-cancer agent and a pharmaceuticallyacceptable adjuvant.

In another alternate preferred embodiment, these methods are useful forinhibiting inflammation and inflammatory diseases in a mammal. Suchmethods are useful in treating or preventing diseases, including,osteoarthritis, acute pancreatitis, chronic pancreatitis, asthma andadult respiratory distress syndrome.

These methods comprise the step of administering to the mammal acomposition comprising a compound of any of formulae I-IX, and apharmaceutically acceptable adjuvant. In a preferred embodiment, thisparticular method comprises the additional step of administering to saidmammal a composition comprising an antiinflammatory agent and apharmaceutically acceptable adjuvant.

In order that this invention be more fully understood, the followingexamples are set forth. These examples are for the purpose ofillustration only and are not to be construed as limiting the scope ofthe invention in any way.

General Materials and Methods

All temperatures are recorded in degrees Celsius. Thin layerchromatography (TLC) was carried out using 0.25 mm thick E. Merck silicagel 60 F₂₅₄ plates and elution with the indicated solvent system.Detection of the compounds was carried out by treating the plate with anappropriate visualizing agent, such as 10% solution of phosphomolybdicacid in ethanol or a 0.1% solution of ninhydrin in ethanol, followed byheating, and/or by exposure to UV light or iodine vapors whenappropriate. Analytical HPLC was carried out using a RaininMycrosorb-MV, 5 μ Cyano reverse phase column, 3.9 mm×150 mm, with a flowrate of 1.0 mL/minute and a solvent gradient of 5-100% acetonitrile(0.1% TFA) in water (0.1% TFA). HPLC retention times were recorded inminutes. NMR spectral data was acquired using a Bruker AMX500 in theindicated solvent.

The IMP dehydrogenase HPLC assay follows our standard conditions for theenzymatic production of XMP and NADH from IMP and NAD, but utilizes highpressure liquid chromatography on a C18 column with ion pairing reagentsto separate all four components. The extent of reaction is thendetermined from the resulting product peak areas. This assay isparticularly useful for determining the inhibition profiles of compoundswhich have significant absorbance in the UV-visible region between 290and 340 nM.

The reaction mixture typically contains 0.1 M KPi; pH 8.0, 0.1M KCl, 0.5mM EDTA, 2 mM DTT, and 0.2 mM each of IMP and NAD. This solution isincubated at 37° C. for 10 minutes. The reaction is started by theaddition of enzyme to a final concentration of 20 to 100 nM, and isallowed to proceed for 10 minutes. After the allotted time, the reactionis quenched by the addition of mycophenolic acid to a finalconcentration of 0.01 mM.

The extent of conversion is monitored by HPLC using a Rainin MicrosorbODS column C18-200 of dimensions 4.6×10 mm and a solvent systemcontaining tetrabutylammonium sulfate (5 mM) in 0.1 M KPi pH 6.0 with a0-30% methanol gradient over 15 minutes. A similar solvent system hasbeen used previously for the purification of halo-IMP derivatives. [L.C. Antionio and J. C. Wu, Biochemistry, 33, 1753-1759 (1994).] AUV-monitor set at 254 nM is used to detect the four components, and theproduct peaks are integrated to determine the extent of conversion ofthe substrates.

For the analysis of inhibitors, the compound in question is dissolved inDMSO to a final concentration of 20 mM and added to the initial assaymixture at the desired concentration in a volume of 2-5% (v/v). Thereaction is started by the addition of enzyme and after 10 minutes isquenched as above. After HPLC analysis, the product areas are used todetermine the extent of conversion relative to a control assaycontaining only DMSO and no test compound. IC50 or Ki values aredetermined from non linear least squares fitting of conversion vsconcentration curves to the tight-binding equations of Henderson. [P. J.F. Henderson, Biochem. J., 127, 321 (1972).]

We have measured the inhibition constants of each compound against IMPDHusing an adaptation of the method first reported by Magasanik. [B.Magasanik, H. S. Moyed, and L. B. Gehring J. Biol. Chem., 226, p.339(1957)].

Insofar as compounds of formulae I-IX are able to inhibit IMPDH, theyare of evident clinical utility for the treatment of IMPDH mediateddisease. These tests are predictive of the compounds ability to inhibitIMPDH in vivo.

Experimental Section

Synthesis of Representative Examples:

EXAMPLE 1

To a solution of 25 mg (156 μmole) 4-(5-oxazolyl)-aniline in 250 μLCH₂Cl₂ was added 50 μL (400 μmole) of benzyl isocyanate at ambienttemperature. After stirring overnight, 1 was isolated in pure form byfiltration with a 3:1 hexanes/CH₂Cl₂ rinse in a yield of 21 mg (46%). ¹HNMR (500 MHz, CDCl₃) δ 7.86 (s), 7.55 (d), 7.38 (d), 7.22-7.35 (m), 6.39(s), 5.0 (br s), 4.43 (s). R_(f) 0.30 (5% MeOH/CH₂Cl₂).

EXAMPLE 2

To a solution of glacial acetic acid (46 mL), acetic anhydride (46 mL,485 mmole) and 2-chloro-4-nitrotoluene (5 g, 29.1 mmole) at 0° C. wasadded conc. H₂SO₄ (6.9 mL) in a dropwise fashion. Upon completeaddition, CrO₃ (8.08 g, 80.8 mmole) was added portion-wise over 60 mins.Following an additional 15 mins of stirring at 0° C., the reactionmixture was poured over ice and the resulting precipitate was isolatedby filtration, rinsing with cold H₂O. Purification by flashchromatography, eluting with a gradient of 15-50% EtOAc in hexanes,provided 2.02 g (24%, 40% based on recovered starting material) B1 as awhite solid. The ¹H NMR was consistent with that of the desiredstructure.

Compound B1 was dissolved in 1:1 ethanol/water (20 mL), treated withconc. H₂SO₄ (2 mL) and refluxed for 1 hour. Upon cooling to ambienttemperature, the reaction was extracted 3×'s with diethyl ether. Theethereal solution was washed twice with water, dried over Na₂SO₄ andconcentrated in vacuo to yield a yellow solid. Purified product wasobtained through two recrystallizations from hot Et₂O/hexanes, yielding620 mg (47.6%) B2 as a lightly yellowed crystalline solid. The ¹H NMRwas consistent with that of the desired structure.

A mixture of B2 (200 mg, 1.2 mmol), tosylmethyl isocyanide (236 mg, 1.2mmol), and powdered K₂CO₃ (172 mg, 1.2 mmole) in methanol (13 mL) washeated at reflux for 90 minutes and then stirred overnight at ambienttemperature. Upon concentration to dryness, the mixture was partitionedbetween CH₂Cl₂ and water. The organics were separated, washed with 0.5NHCl, water and brine and then dried over Na₂SO₄. The solvent was removedin vacuo to provide a crude yellow solid. Purified product B3 wasobtained through flash chromatography, eluting with a gradient of 0-2.5%CH₃OH in CH₂Cl₂, and recrystallization (CH₂Cl₂/hexanes) in a yield of3.3 g (68%) as a light yellow crystalline solid. The ¹H NMR wasconsistent with that of the desired structure.

A solution of B3 (150 mg, 0.67 mmole) in ethanol (7.5 mL) was treatedwith SnCl₂.2H₂O (excess; ca. 5 equivalents) and heated at reflux for 30minutes. The mixture was cooled to ambient temperature, diluted withdiethyl ether and partitioned with 2N NaOH. The organics were separated,washed with water and brine, dried over Na₂SO₄ and concentrated invacuo. Purified product B4 was obtained through flash chromatography,eluting with a gradient of 0-0.5% CH₃OH in CH₂Cl₂, in a yield of 54 mg(41.5%) as a light yellow oil. The ¹H NMR was consistent with that ofthe desired structure.

To a solution of 20 mg (103 μmole) B4 in 1 mL CH₂Cl₂ was added 20 μLm-tolylisocyanate at ambient temperature. After stirring overnight, 43was isolated in pure form by filtration with an EtOAc/hexanes rinse in ayield of 25 mg (74%). ¹H NMR (500 MHz, d₆-DMSO) δ 9.06 (s), 8.73 (s),8.50 (s), 7.89 (s), 7.73 (d), 7.67 (s), 7.42 (d), 7.31 (s), 7.23 (d),7.18 (t), 6.82 (d), 2.27 (s). R_(f) 0.28 (5% MeOH/CH₂Cl₂).

EXAMPLE 3

C1 (8.14 g, 51%) was prepared from 2-methyl-5-nitroanisole (10.0 g, 60mmole) in a fashion directly analogous to the preparation of B1 asdescribed above. The ¹H NMR was consistent with that of the desiredstructure.

A stirred suspension of C1 (81.94 g, 307 mmole) in dioxane (100 mL) wastreated with concentrated HCl (20 mL) and heated at reflux overnight.Upon cooling to ambient temperature, the product C2 precipitated as alight yellow crystalline solid in a yield of 40.65 g (73.1%). Thefiltrate was concentrated to a volume of ca. 80 mL and a second crop ofproduct crystals was driven from solution by the addition of hexanes,yielding 8.91 g (16.0%). Both batches were identical by ¹H NMR and TLCanalysis and were consistent with that of the desired material. Thetotal yield of C2 was 49.56 g (89.1%).

A solution of C2 (456 mg, 2.51 mmole), tosylmethyl isocyanide (490 mg,2.51 mmole) and K₂CO₃ (347 mg, 251 mmole) were dissolved in methanol andheated to reflux for 1.5 hours. The product mixture was thenconcentrated in vacuo, redissolved in CH₂Cl2, washed with water andbrine, dried over Na₂SO₄ and again concentrated in vacuo. Purifiedproduct C3 was obtained through recrystallization (Et₂O/hexanes) toyield 375 mg (68%). The ¹H NMR was consistent with that of the desiredstructure.

A solution of C3 (4.214 g, 19.1 mmole) in EtOAc (150 mL) was treatedwith 10%Pd/C (1.05 g, 25 wt. % of C3) and subjected to 40 psi H₂(g)(Parr Hydrogenation Apparatus) overnight. The reaction mixture wasfiltered and concentrated in vacuo. Pure product C4 was obtained throughflash chromatography, eluting with a gradient of 30-40% EtOAc/hexanes,in a yield of 3.4 g (93%). The ¹H NMR was consistent with that of thedesired structure.

To a solution of C4 (25 mg, 0.131 mmole) in CH₂Cl₂ (1 mL) was added tollisocyanate (25 μL, 0.197 mmole) at ambient temperature. After stirringovernight, 56 was isolated in pure form by filtration with a CH₂Cl₂rinse in a yield of 42 mg (74%). ¹H NMR (500 MHz, d₆-DMSO) δ 8.87 (s),8.64 (s), 8.37 (s), 7.60 (d), 7.46 (d), 7.42 (s), 7.33 (s), 7.23 (d),7.16-7.19 (t), 7.05 (dd), 6.80 (d), 3.92 (s), 2.28 (s). R_(f) 0.46 (5%MeOH/CH₂Cl₂).

EXAMPLE 4

To a solution of C4 (75 mg, 0.394 mmole) in dichloroethane (5 mL) wasadded 3-nitrophenyl isocyanate (97 mg, 0.591 mmole) at ambienttemperature. After stirring overnight, Dl was isolated in pure form byfiltration with a CH₂Cl₂ rinse in a yield of 110.3 mg (79%). The ¹H NMRwas consistent with that of the desired structure.

To a stirred suspension of Dl (95 mg, 0.268 mmole) in EtOH (20 mL) wasadded SnCl₂.2H₂O (302 mg, 1.34 mmole). The reaction mixture was broughtto reflux, at which time dissolution occurred, for 1.5 hours. Thesolution was cooled to ambient temperature, diluted with EtOAc, washedwith 2N NaOH and brine, dried (Na₂SO₄) and concentrated in vacuo. Pureproduct 59 was obtained through flash chromatography (eluting with agradient of 2.5-5% MeOH in CH₂Cl₂), followed by selectivecrystallization of the desired material from slightly impure fractionsin a yield of 15.7 mg (18%). ¹H NMR (500 MHz, d₆-DMSO) δ 8.83 (s), 8.44(s), 8.35 (s), 7.59 (d), 7.48 (d), 7.40 (s), 6.97-7.04 (dd), 6.86-6.92(t), 6.83 (d), 6.54 (dd), 6.20 (dd), 5.05 (brs), 3.92 (s). R_(f) 0.20(5% MeOH/CH₂Cl₂).

EXAMPLE 5

A solution of 3-aminobenzylamine (826 mg, 6.87 mmole) and triethylamine(2.39 mL, 17.18 mmole) was treated with di-t-butyldicarbonate (1.50 g,6.87 mmole) and the mixture was stirred at ambient temperature for 2hours. The reaction was then diluted with CH₂Cl₂, washed withNaHCO₃(aq), water and brine, dried (Na₂SO₄) and concentrated in vacuo.Pure E1 was obtained by flash chromatography, eluting with 25% EtOAc inhexanes in a yield of 200 mg (46%). The ¹H NMR was consistent with thatof the desired structure.

A solution of C4 (150 mg, 0.789 mmole) and 1,1-dicarbonylimidiazole (160mg, 0.986 mmole) were combined in THF (5 mL) and stirred for 6 hours atambient temperature. The precipitation of imidazole was noted. To thiswas then added E1 (351 mg, 1.58 mmole) and N,N-dimethylaminopyridine (97mg, 0.789 mmole) and the mixture was refluxed overnight, resulting in ahomogenous solution. Upon cooling to ambient temperature, the reactionwas diluted with EtOAc (20 mL), washed with KHSO₄(aq), water, and brine,dried (MgSO₄) and concentrated. Pure 113 was obtained through flashchromatography, eluting with a gradient of 20-30-35% acetone in hexanesin a yield of 164 mg (47%). ¹H NMR (500 MHz, d₆-DMSO) δ 8.90 (s), 8.75(s), 8.38 (s), 7.60 (d), 7.51 (s), 7.3-7.46 (m), 7.21-7.27 (t), 7.05(dd), 6.87 (d), 4.12 (d), 3.93 (s), 1.44 (s). R_(f) 0.21 (5%MeOH/CH₂Cl₂).

EXAMPLE 6

A solution of 3-chloro-4-cyanoaniline (500 mg, 7.76 mmole) andm-tolylisocyanate (1.0 mL, 3.17 mmole) in CH₂Cl₂ (3 mL) was stirredovernight at ambient temperature. The reaction mixture was concentratedand pure 70 was obtained through MPLC, eluting with 1% MeOH in CH₂Cl₂,in a yield of 285 mg (31%). ¹H NMR (50 MHz, d₆-DMSO) δ 9.36 (s), 8.88(s), 7.94 (s), 7.83 (d), 7.44 (d), 7.30 (s), 7.24 (d), 7.15-7.20 (t),6.82 (d), 2.29 (s). R_(f) 0.36 (5% MeOH/CH₂Cl₂).

EXAMPLE 7

To a solution of 3,4,5-trimethoxyacetophenone (9.2 g, 43.4 mmol) inpyridine (35 mL) was added selenium dioxide (6.3 g, 56.7 mmol) and theresulting solution was heated at reflux overnight. The reaction mixturewas cooled to ambient temperature, filtered through celite andconcentrated to yield a dark brown oil which was dissolved into ethylacetate and washed with 1.0 N HCl and then with saturated NaHCO₃. Thebasic aqueous layer was diluted with ether and acidified withconcentrated HCl. The layers were separated and the organic phase waswashed with brine and then dried (Na₂SO₄) to give 8.4 g of a dark yellowsolid. Recrystallization of this material from ethyl acetate-hexane thengave G1 (6.8 g) as a pale yellow solid. The ¹H NMR was consistent withthat of the desired structure.

A mixture of 59 (64 mg, 0.20 mmole), G1 (300 mg, 1.20 mmole) and EDC(300 mg, 1.6 mmole) in THF (5 mL) was stirred overnight at ambienttemperature. The reaction was diluted with EtOAc (150 mL), washed withwater, dried (MgSO₄) and concentrated in vacuo. Pure 108 was obtainedthrough MPLC, eluting with a gradient system of 0-1%MeOH in CH₂Cl₂, in ayield of 37.4 mg (35%). ¹H NMR (500 MHz, d₆-DMSO) δ 9.83 (s), 8.23 (s),8.18 (s), 7.65 (s), 7.61 (s), 7.35 (d), 7.33 (s), 7.29 (s), 7.27 (s),7.11 (s), 7.06-7.10 (t), 6.94-6.99 (t), 6.52 (d)3.68 (s), 3.63 (s), 3.61(s). R_(f) 0.26 (5% MeOH/CH₂Cl₂).

EXAMPLE 8

A solution of 59 (300 mg, 1.58 mmole) and m-toll isothiocyanate (2.0 mL,14.7 mmole) in CH₂Cl₂ (5 mL) was stirred at ambient temperatureovernight. To drive the reaction to completion, additional m-tollisothiocyanate (1.0 mL, 7.4 mmole) was added and the mixture was heatedto reflux for 3 hours. The reaction was concentrated in vacuo and 115was obtained in pure form through MPLC, eluting with 0-5% EtOAc inCH₂Cl₂, in a yield of 210 mg (39%). ¹H NMR (500 MHz, d₆-DMSO) δ 7.90(s), 7.89 (s), 7.82 (s), 7.75 (d), 7.64 (s), 7.44 (s), 7.32-7.37 (t),7.27 (s), 7.13-7.21 (m), 6.91 (dd), 3.98 (s), 2.40 (s). R_(f) 0.36 (5%MeOH/CH₂Cl₂).

EXAMPLE 9

A solution of nitroaniline (1.0 g, 7.13 mmole) in CH₂Cl₂ (25 mL) wastreated with pyridine (2.9 mL, 36 mmole) and trifluoroacetic anhydride(5 mL, 36 mmole) and stirred at ambient temperature for 3 hours. Thereaction was diluted further with CH₂Cl2, washed with 1N HCl and brine,dried (MgSO₄) and concentrated in vacuo to yield I1 (1.61 g, 95%) as awhite solid. The ¹H NMR was consistent with that of the desiredstructure.

To a slurry of NaH (60% oil dispersion; 34 mg, 1.42 mmole) in THF (10mL) at 0° C. was added a solution of I1 (200 mg, 0.85 mmole) in THF (10mL) and the mixture stirred for 1 hour. To this was added methyl iodide(100 μL, 1.7 mmole) and the mixture was stirred overnight at ambienttemperature. The reaction was poured into water and extracted withEtOAc. The organics were separated, dried (MgSO₄) and concentrated invacuo. Pure I2 was obtained through flash chromatography, eluting with5% EtOAc in hexanes, in a yield of 163 mg (66%) as a yellow solid. The¹H NMR was consistent with that of the desired structure.

A solution of I2 (163 mg, 0.66 mmole) in ethanol (5 mL) was treated withPd/C (20 mg) and subjected to H₂ (1 atm.) for 3 hours. The reaction wasfiltered and concentrated in vacuo to yield I3 (120 mg, 84%) as a waxysolid. The ¹H NMR was consistent with that of the desired structure.

To a solution of triphosgene (31 mg, 0.104 mmole) in dichloroethane (1mL) was added in a dropwise fashion a solution of B4 (50 mg, 0.260mmole) and diisopropylethylamine (67 mg, 518 mmole) in dichloroethane (5mL). The reaction mixture was stirred for an additional 1 hour atambient temperature, treated with I3 (50 mg, 0.230 mmole) and stirredovernight. The entire reaction mixture was subjected to flashchromatography, eluting with 1% MeOH in CH₂Cl₂, to provide pure 97 in ayield of 8 mg (7%). ¹H NMR (500 MHz, d₆-DMSO) δ 9.20 (s), 8.98 (s), 8.39(s), 7.67 (s), 7.63 (d), 7.48 (s), 7.38-7.45 (m), 7.04-7.10 (t), 3.95(s), 3.31 (s). R_(f) 0.37 (5% MeOH/CH₂Cl₂).

EXAMPLE 10

A solution of 59 (50 mg, 0.154 mmole) and triethylamine (31 mg, 0.308mmole) in DMF (0.5 mL) was treated in a dropwise fashion withphenylacetyl chloride (25 mg, 0.169 mmole) and the reaction stirredovernight at ambient temperature. The mixture was diluted with CH₂Cl₂,washed with NaHCO₃(aq) and water, dried over MgSO₄ and concentrated invacuo. Pure 111 was isolated by flash chromatography, eluting with 2%MeOH in CH₂Cl2, in a yield of 42 mg (62%). ¹H NMR (500 MHz, d₆-DMSO) δ10.20 (s), 8.90 (s), 8.79 (s), 8.39 (s), 7.88 (s), 7.63 (d), 7.53 (d),7.44 (s), 7.25-7.40 (m), 7.22 (t), 7.14 (d), 7.05 (dd), 3.96 (s), 3.66(s). R_(f) 0.31 (5% MeOH/CH₂Cl₂)

EXAMPLE 11

A solution of 2-methyl-5-nitrobenzoic acid (15 g, 82.8 mmole) in DMF (75mL) was treated with methyl iodide (6.7 mL, 107.64 mmole) followed bypowdered K₂CO3 (17.2 g, 124.2 mmole) (extreme exotherm) and thesuspension stirred at ambient temperature overnight. The reactionmixture was partitioned between EtOAc and water, the organics separatedand washed with water and brine, dried (Na₂SO₄) and concentrated invacuo to yield K1 (15.86 g, 98%) in pure form as an off-white solid. The¹H NMR was consistent with that of the desired structure.

K2 (4.09 g, 16.2%) was prepared from K1 (15.86 g, 81.3 mmole) in afashion analogous to the preparation of B1 as described above. The ¹HNMR was consistent with that of the desired structure.

A solution of K2 (2.5 g, 8.03 mmole) in dioxane (10 mL) was treated withconc. HCl (0.5 mL) and the mixture was heated to reflux for 2 hours.Additional conc. HCl (0.5 mL) was added and the reaction refluxed for 3hours longer. The mixture was diluted with EtOAc, washed with water andbrine, dried (Na₂SO₄) and concentrated in vacuo. Pure K3 was obtainedthrough flash chromatography, eluting with a gradient of 20-30-50% Et₂Oin hexanes, in a yield of 1.14 g (68%). Also isolated was 215 mg (11.8%)of the hydrated aldehyde. The ¹H NMRs were consistent with that of thedesired structures.

A solution of K3 (300 mg, 1.43 mmole) in benzene (5 mL) was treated with1,3-propane diol (114 μL, 1.573 mmole) and p-TsOH.H₂O (27 mg, 0.14mmole) and the mixture was refluxed with Dean-Stark removal of water for4.5 hours. The reaction was cooled to ambient temperature, partitionedbetween EtOAc and dilute NaHCO₃, the organics separated, washed withbrine, dried (Na₂SO₄) and concentrated in vacuo. Pure K4 was obtainedthrough flash chromatography, eluting with a gradient of 20-25% Et₂O inhexanes, in a yield of 324 mg (84.5%) as an off-white crystalline solid.The ¹H NMR was consistent with that of the desired structure.

A solution of K4 (289 mg, 1.08 mmole) in THF (5 mL) at 0° C. was treateddropwise with a solution of DIBAL (1.0 M in CH₂Cl₂; 2.7 mL, 2.7 mmole)and stirred for 40 minutes. The reaction was quenched by addition ofsaturated Rochelle's salt solution (10 mL), diluted with EtOAc andstirred for 30 minutes. The organics were collected, washed with brine,dried (Na₂SO₄) and concentrated in vacuo to give 250 mg (97%) of K5 as awhite crystalline solid. The ¹H NMR was consistent with that of thedesired structure.

A solution of K5 (250 mg, 1.05 mmole) in CH₂Cl₂ (4 mL) at 0° C. wastreated with pyridine (110 μL, 1.37 mmole), benzoyl chloride (146 μL,1.26 mmole) and 4-DMAP (catalytic), and stirred at ambient temperatureovernight. The reaction mixture was diluted with CH₂Cl₂, washed with0.5N HCl, water and brine, dried (Na₂SO₄) and concentrated in vacuo.Pure K6 was obtained through flash chromatography, eluting with 10%EtOAc in hexanes, in a yield of 340 mg (99%) as a white solid. The ¹HNMR was consistent with that of the desired structure.

A solution of K6 (326 mg, 0.99 mmole) in dioxane (7 mL) was treated with2.0 N HCl (5 mL) and the mixture heated at 80° C. overnight. Thereaction mixture was diluted with EtOAc and washed with saturatedNaHCO₃(aq), water and brine, dried (Na₂SO₄) and concentrated in vacuo.Pure K7 was obtained through flash chromatography, eluting with 30% Et₂Oin hexanes, in a yield of 208 mg (77.5%) as a white solid. The ¹H NMRwas consistent with that of the desired structure.

A solution of K7 (208 mg, 0.729 mmole) in MeOH (6 mL) was treated withK₂CO₃ (101 mg, 0.765 mmole) and TosMIC (149 mg, 0.765 mmole) and thesolution heated at 60° C. for one hour. The reaction was concentrated invacuo, redissolved in CH₂Cl₂ and washed with 1.0N NaOH (diluted withsaturated NaHCO₃). The aqueous portion was back-extracted with CH₂Cl2,the organics combined and washed with water and brine, dried (Na₂SO₄)and concentrated in vacuo. Pure K8 was obtained through flashchromatography, eluting with a gradient of 10-50% acetone in hexanes, ina yield of 70 mg (44%). The ¹H NMR was consistent with that of thedesired structure.

A solution of K8 (70 mg, 0.318) in acetic anhydride (1.5 mL) andpyridine (1.0 mL) was treated with 4-DMAP (catalytic) and stirred atambient temperature for 3 hours. The mixture was diluted with CH₂Cl₂,washed with 1.0N HCl, water and brine, dried (Na₂SO₄) and concentratedin vacuo to provide K9 in a yield of 82 mg (98%) as a pale yellow solid.The ¹H NMR was consistent with that of the desired structure.

A solution of K9 (80 mg, 0.305 mmole) in dry EtOH (4 mL) was treatedwith SnCl₂.2H₂O (241 mg, 1.07 mmole) and the mixture heated at 60° C.for 50 minutes. The reaction was diluted with EtOAc, washed withsaturated NaHCO₃, water and brine, dried (Na₂SO₄) and concentrated invacuo. Pure K10 was obtained through flash chromatography, eluting witha gradient of 20-30% acetone in hexanes, in a yield of 52 mg (73.4%) asa pale yellow oil. The ¹H NMR was consistent with that of the desiredstructure.

A solution of K10 (52 mg, 0.224 mmole) in dichloroethane (2 mL) wastreated with m-tolyl isocyanate (43 μL, 0.336 mmole) and stirredovernight at ambient temperature. The mixture was diluted withCH₂Cl₂:hexanes (2:1), filtered and rinsed with the same solvent systemto provide K11 (67 mg, 82%) as a white solid. The ¹H NMR was consistentwith that of the desired structure.

A solution of K11 (33 mg, 0.09 mmole) in MeOH (2 mL) was treated with1.0N NaOH (135 μL, 0.135 mmole) and stirred at ambient temperature for1.5 hours. The reaction was neutralized by addition of 1.0N HCl (135 μL)and concentrated in vacuo. The white solid was rinsed with water andCH₂Cl₂:hexanes (2:1) and dried in vacuo to provide 102 (20 mg, 68%) as awhite solid. ¹H NMR (500 MHz, d₆-DMSO) δ 9.29 (s), 9.00 (s), 8.42 (s),7.69 (s), 7.55 (m), 7.37 (s), 7.33 (s), 7.27 (d), 7.16 (t), 6.80 (d),5.39 (t), 4.58 (s), 2.28 (s). R_(f) 0.13 (1:1 hexanes/acetone).

EXAMPLE 12 Synthesis of Compound 106

A solution of C4 (50 mg, 0.263 mmole) in THF (2 mL) was treated with CDI(53 mg, 0.330 mmole) and stirred at ambient temperature for 4 hours. Tothis was added 1-acetyl-6-aminoindole (93 mg, 0.526 mmole, SigmaChemical Co.) and 4-DMAP (35 mg, 0.289 mmole) and the mixture refluxedovernight. Diluted with EtOAc (100 mL), washed with 5% KHSO₄, water andbrine, dried (Na₂SO₄) and concentrated in vacuo. Redissolved in EtOAcand filtered to removed insoluble materials and reconcentrated in vacuo.Pure 106 was obtained through flash chromatography, eluting with agradient of 50-60% acetone in hexanes, in a yield of 37 mg (36%) as awhite solid. ¹H NMR (500 MHz, d₆-DMSO) δ 8.79 (s), 8.74 (s), 8.37 (s),8.11 (s), 7.62 (d), 7.47 (s), 7.43 (s), 7.30 (d), 7.13 (d), 7.14 (d),4.11 (t), 3.94 (s), 3.07 (t), 2.17 (s). R_(f) 0.14 (1:1hexanes/acetone).

EXAMPLE 13

A suspension of 113 (from Example 5) (250 mg, 5.76 mmol) in CH₂Cl₂ (1mL) was treated in a dropwise fashion at ambient temperature withseveral equivalents of trifluoroacetic acid and stirred for 90 min. Theresulting solution was stripped in vacuo and tritrated with CH₂Cl₂ andmethanol. Pure product 168 was isolated by filtration in a yield of 258mg (99%). The ¹H NMR was consistent with that of the desired product.

A suspension of 168 (250 mg, 0.55 mmol) in 21 mL of CH₂Cl₂/DMF (20:1 byvolume) was treated with triethyl amine (193 μL, 1.38 mmol) and stirredat ambient temperature until homogeneity was reached. The solution wascooled to 0 C, treated with (S) 3-tetrahydrofuranyl-N-oxysuccinimidylcarbonate (635 mg, 0.608 mmol) and allowed to stir overnight withwarming to ambient temperature. The mixture was poured into ethylacetate (500 mL), washed with NaHCO₃(aq)(2×), water (2×), and brine(1×),dried over Na₂SO₄ and stripped in vacuo. Pure product 120 was isolatedby tritration (30 mL CH₂Cl₂, 100 mL ether) in a yield of 212 mg (85%).The ¹H NMR was consistent with that of the desired product.

EXAMPLE 14 IMPDH Activity Inhibition Assay

We measured the inhibition constants of the compounds listed in TableIII utilizing the following protocol:

IMP dehydrogenase activity was assayed following an adaptation of themethod first reported by Magasanik. [Magasanik, B. Moyed, H. S. andGehring L. B. (1957) J. Biol. Chem. 226, 339]. Enzyme activity wasmeasured spectrophotometrically, by monitoring the increase inabsorbance at 340 nm due to the formation of NADH (ε340 is 6220 M⁻¹cm⁻¹) . The reaction mixture contained 0.1 M Tris pH 8.0, 0.1 M KCl, 3mM EDTA, 2 mM DTT, 0.1 M IMP and enzyme (IMPDH human type II) at aconcentration of 15 to 50 nM. This solution is incubated at 37° C. for10 minutes. The reaction is started by adding NAD to a finalconcentration of 0.1M and the initial rate is measured by following thelinear increase in absorbance at 340 nm for 10 minutes. For reading in astandard spectrophotometer (path length 1 cm) the final volume in thecuvette is 1.0 ml. The assay has also been adapted to a 96 wellmicrotiter plate format; in this case the concentrations of all thereagents remain the same and the final volume is decreased to 200 μl.

For the analysis of inhibitors, the compound in question is dissolved inDMSO to a final concentration of 20 mM and added to the initial assaymixture for preincubation with the enzyme at a final volume of 2-5%(v/v). The reaction is started by the addition of NAD, and the initialrates measured as above. K_(i) determinations are made by measuring theinitial velocities in the presence of varying amounts of inhibitor andfitting the data using the tight-binding equations of Henderson(Henderson, P. J. F. (1972) Biochem. J. 127, 321].

These results are shown in Table III. K_(i) values are expressed in nM.Category “A” indicates 0.01 to 50 nm activity, category “B” indicates51-1000 nm activity, category “C” indicates 1001 to 10,000 nm activity,category “D” indicates greater than 10,000 nm activity. The designation“ND” is used where a given compound was not tested.

TABLE III Cmpd K_(i) # (nM) 1 C 2 C 3 B 4 D 5 C 6 C 7 B 8 C 9 C 10 C 11C 12 C 13 C 14 C 15 C 16 C 17 B 18 C 19 C 20 C 21 C 22 C 23 C 24 B 25 C26 C 27 C 28 C 29 D 30 C 31 D 32 D 33 D 34 C 35 C 36 C 37 C 38 D 39 D 40C 41 C 42 B 43 B 44 — 45 C 46 B 47 B 48 C 49 C 50 D 51 D 52 C 53 C 54 C55 A 56 B 57 B 58 C 59 A 60 B 61 D 62 C 63 C 64 B 65 B 66 C 67 C 68 B 69B 70 C 71 C 72 C 73 B 74 B 75 B 76 C 77 B 78 B 79 B 80 C 81 C 82 C 83 B84 B 85 B 86 C 87 D 88 C 89 C 90 C 91 C 92 C 93 A 94 B 95 C 96 B 97 A 98B 99 A 100 B 101 C 102 C 103 C 104 C 105 B 106 B 107 A 108 B 109 B 110 B111 A 112 B 113 A 114 B 115 B 116 A 117 B 118 C 119 A 120 A 121 A 122 A123 A 124 A 125 A 126 A 127 A 128 A 129 A 130 A 131 A 132 A 133 A 134 A135 A 136 A 137 B 138 A 139 B 140 A 141 A 142 A 143 B 144 B 145 A 146 A147 A 148 A 149 A 150 A 151 B 152 B 153 A 154 A 155 A 156 A 157 B 158 B159 A 160 A 161 A 162 A 163 B 164 B 165 A 166 D 167 B 168 B

EXAMPLE 15 Anti-Viral Assays

The anti-viral efficacy of compounds may be evaluated in various invitro and in vivo assays. For example, compounds may be tested in invitro viral replication assays. In vitro assays may employ whole cellsor isolated cellular components. In vivo assays include animal modelsfor viral diseases. Examples of such animal models include, but are notlimited to, rodent models for HBV or HCV infection, the Woodchuck modelfor HBV infection, and chimpanzee model for HCV infection.

While we have described a number of embodiments of this invention, it isapparent that our basic constructions may be altered to provide otherembodiments which utilize the products and methods of this invention.Therefore, it will be appreciated that the scope of this invention is tobe defined by the appended claims, rather than by the specificembodiments which have been presented by way of example.

We claim:
 1. A composition comprising: a. a compound having the formula:

and b. a pharmaceutically acceptable adjuvant.
 2. The compositionaccording to claim 1, additionally comprising an agent selected from animmunosuppressant, an anti-cancer agent, an anti-viral agent,antiinflammatory agent, antifungal agent, antibiotic, or ananti-vascular hyperproliferation agent.
 3. The composition according toclaim 2, wherein said additional agent is an immunosuppressant.
 4. Thecomposition according to claim 2, wherein said additional agent is ananti-viral agent.
 5. The composition according to claim 2, wherein saidadditional agent is an anti-vascular hyperproliferation agent.
 6. Thecomposition according to claim 2, wherein said additional agent is ananti-cancer agent.
 7. The composition according to claim 2, wherein saidadditional agent is an antiinflammatory agent.
 8. The compositionaccording to claim 2, wherein said additional agent is an anti-fungalagent.
 9. The composition according to claim 2, wherein said additionalagent is an antibiotic.
 10. A method of treating a disease caused by HBVor HCV comprising the step of administering to said mammal a compositionaccording to any of claims 1, 2 or
 4. 11. The method according claim 10,wherein the viral disease is caused by HBV.
 12. The method accordingclaim 10, wherein the viral disease is caused by HCV.